Pharmacology Biochemistry & Behavior, Vol. 4, pp. 39--44. Copyright © 1976 by ANKHO International Inc. All rights of reproduction in any form reserved. Printed in the U.S.A.

Effect of Stress on the Uptake of aH-Norepinephrine into Rat Myocardium' J. R. BASSETT AND K. D. CAIRNCROSS School o f Biological Sciences, Macquarie University, North Ryde, N.S. W., 2113, Australia (Received 31 March 1975) BASSETT, J. R. AND K. D. CAIRNCROSS. Effect of stress on the uptake of JH-norepinephrine into rat myocardium. PHARMAC. BIOCHEM. BEHAV. 4(1) 39-44, 1976. - Spontaneously beating atria from rats previously exposed to irregular, signalled footshock were incubated with a H-norepinephrine. A significant reduction in the uptake and retention of radioactivity was found in the atria from stressed animals compared with unstressed controls. A kinetic study of the uptake process in both the stressed and control groups showed a similar Km value but a significantly different Vma x. It was concluded that the enhanced myocardial sensitivity to catecholamines previously reported can be explained in part, on the basis of an inhibition of neuronal uptake. Stress

Catecholamine uptake

Uptake inhibition

Corticosteroids

kinetic constants were used to identify the uptake process most affected by the stress procedure. The contribution of 3H-norepinephrine to the total radioactivity obtained from homogenated rat atria was determined using chromatographic separation. Iversen [ 11 ] suggested that circulating transmitter removed by Uptakes may subsequently suffer extraneuronal metabolism. Any changes in the proportions of unchanged norepinephrine or its metabolites may indicate inhibition of metabolic enzymes which, together with the inhibition of uptake, may play a role in the stress induced enhanced myocardial sensitivity.

BASSETT and Cairncross [1] reported that exposure of male rats to irregular signalled footshock from which the animal could e~cape resulted in an enhanced myocardial sensitivity to both norepinephrine and epinephrine. On the basis of changes in the endogenous levels of norepinephrine and epinephrine following exposure to stress, an inhibition of the uptake of catecholamines into storage sites was proposed as a possible contributing factor to the enhanced myocardial sensitivity [2]. Furthermore it was suggested that such a stress procedure may inhibit neuronal uptake (Uptake I ) as welt as extraneuronal uptake (Uptake 2). To confirm the presence of a stress induced inhibition of uptake the effect of irregular-signalled escape stress on the myocardial accumulation of a H-norepinephrine by spontaneously beating isolated rat atria, was measured following various periods of incubation. In order to clarify which uptake mechanism was affected by stress a kinetic study was undertaken. Kinetic analysis of both the Uptakel and Uptake 2 mechanism show both qualitative and quantitative difference [4, 8, 9, 10]. These differences in kinetic properties act to characterise each uptake system. Uptake1 is operative at low catecholamine concentrations whereas Uptake 2 becomes more apparent at high concentrations. Both processes saturate with increasing external concentration and can be described by MichaelisMenten kinetics. The affinity of (+)-norepinephrine for Uptake2 is 374 times less than its affinity for Uptake1, however with Uptake2, norepinephrine uptake is rapid and is completed in a short time [10]. In this study the initial rates of norepinephrine uptake were measured at various incubation concentrations of norepinephrine. The results were subjected to a Michaelis-Menten analysis from which the kinetic constants K m and Vmax were determined. The

METHOD

Animals Male CSF rats 8 7 - 9 3 days old were used in all experiments. The animals were housed in groups of 3 under conditions of constant temperature and humidity (21 -+ 0.5°C, 46 percent humidity) and subjected to a 12 hr night-day routine (light 8 a.m. - 8p.m.) beginning at least 14 days prior to commencement of experimentation. Food and water were provided ad lib. Both control and stressed rats were housed under identical conditions.

Stress A ppara tus and Pro cedure Animals were placed in automated l-way avoidance boxes (Lafayette Model No. 85200) described in detail by Bassett et al. [3]. An escape platform was made available to the animal by an automated movable partition. A light conditioned stimulus (CS) of 2 W was located on the wall of the grid chamber opposite to the escape platform. The

I The skilled technical assistance of Mrs. Carol Martin is gratefully acknowledged. The work reported in this paper was supported by Grant number D1 73/15017 from the Australian Research Grants Committee. 39

40

BASSETT AND CAIRNCROSS

unconditioned stimulus (UCS) was delivered by a generator-scrambler through the grids as a 2 mA, 50 pulses/sec square wave. Each rat was placed on the escape platform at the commencement of the treatment session. Treatment consisted of 7 CS-UCS exposures randomly placed in the 35 min session. On each trial the CS onset 4 sec before the animal was pushed by the movable partition from the platform onto the grid, which was simultaneous with the onset of the UCS. At this the movable partition immediately retracted and the animal was able to jump from the grid to the re-exposed platform with a minimum latency of 0.3 sec. The UCS was terminated by the return of the animal to the platform. Animals were stressed daily, one session/day, for 4 days. Immediately after completion of the last stress period, animals were sacrificed by cervical dislocation and exsanguinated. The blood was collected in heparinized tubes and centrifuged in order to obtain cell free plasma which was then frozen. Plasma corticosterone levels were determined subsequently by the fluorimetric method of Mattingley [14], which is specific for free 11-hydroxycorticosteroids.

Tissue Preparation The atria were suspended in a 15 ml organ bath containing Krebs-Henseleit solution gassed with 5 percent carbon dioxide in oxygen and maintained at 29°C. Atropine sulphate at a concentration of 2.9 x 10 -~ M was added to the Krebs solution. The inotropic response of the spontaneously beating atria was monitored using an isotonic strain gauge transducer and recorded on a Cardiotrace recorder. Preparations were allowed to equilibrate for 30 min after setting up before commencement of the experiment. Ethylenediamine tetra acetic acid (EDTA), 20 ug/ml, was added to the solution in the organ bath 5 min before the addition of norepinephrine. Cumulative log doseresponse curves for norepinephrine were obtained and the EDs0 was derived from the percentage of the maximal response determined for each curve. Regression lines were fitted to the linear portions of the curves by the method of least squares. Each line was tested for linearity and the EDs 0 calculated using regression coefficients. The EDs 0's from 7 experiments were measured and the mean ED s0 calculated. This concentration of norepinephrine was used in the study of accumulation of radioactivity in the myocardium as a function of the time of incubation.

Accumulation

o f radioactivity as a function o~ time.

Control and stressed atria were incubated with °H-7-1norepinephrine (1 × 10 -s g/ml in organ bath; 5.92 x 10 -s M) for periods of 2, 5, 7, 10, 15 and 20 min. The 3H-norepinephrine (Specific activity 8.7 C i / m m o l e ) w a s obtained chromatographically pure from the Radiochemical Centre, Amersham (England). Each batch of 3H-norepinephrine was standardized. Following incubation with the labelled amine the atria were removed from the organ bath, washed in Krebs-Henseleit solution and blotted, this procedure being repeated 3 times. The tissues were then weighed, and homogenized in 3 ml of 0.4 N perchloric acid containing 10 ug/ml cold norepinephrine. The homogenate was centrifuged at 5,000 rpm for 15 min, then 200 ~1 of the supernatant was added to 15 ml of scintillation solution in a counting vial. The scintillation solution was prepared by mixing 350 ml of ethanol with 650 ml of toluene solution containing 4.0 g/1 of 2,5-diphenyloxazole (PPO) and 0.1 g/1 of 1,4-bis-2-(5-phenyloxazolyl)-benzene

(POPOP). All samples were counted for 10 rain periods in a Packard TRI-CARB liquid scintillation counter. Duplicate samples were taken from each homogenate and the counts obtained from the duplicates were meaned. Background blanks were prepared for each experimental series by setting up atria and performing all procedures detailed above with the exception that no 3H-norepinephrine was added to the incubation fluid. The automatic external standarization ratio technique was used to monitor quenching. Efficiency curves were obtained and all counts were corrected for counting efficiency to give the number of disintegrations/min (DPM). The DPM's due to background activity (Blank) were subtracted from the mean DPM obtained from the duplicates for each homogenate. Total tissue radioactivity was then calculated at DPM/mg of atrial tissue after correcting for the water content of the atria. The accumulation of radioactivity in atria from both stressed and control animals after various incubation periods was compared using unpaired t tests.

Initial rate o f norepinephrine uptake at various incubation concentrations. The method was essentially the same as that described above, except that in this series of experiments the incubation time was fixed at 5 min and the concentration of 3 H-7-norepinephrine (specific activity 7.8 Ci/mmole) was varied. At high concentrations cold 1-norepinephrine was added to the bath to make up the required concentration. The DPM measured was corrected accordingly. Results were expressed as DPM/mg/min. In order to see if the norepinephrine uptake obeyed Michaelis-Menten kinetics, the initial rate of norepinephrine uptake at the various concentrations of norepinephrine was plotted in the form S/v against S (S = incubation concentration of norepinephrine (molar bath concentration), v = initial rate of norepinephrine uptake (DPM/mg/min). From the slopes and intercepts of these graphs the kinetic constants Vma x and Km were determined [ 8 ].

Chromatographic separation o f total radioactivity into its component parts. Atria from control and stressed animals were incubated with 7.96 × 10-s M (1.35 × I0 -s g/ml) 3 H-7-1-norepinephrine for 5 min. The atria were then washed, blotted and weighed as described above, then homogenized in 3 ml of 75 percent ethanol containing 0.1 mg of each of the following non-radioactive substances; 1-norepinephrine (NA), normetanephrine (NM), 3-methoxy-4-hydroxymandelic acid (MHMA), 3,4-dihydroxymandelic acid (DHMA), 3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4 dihydroxyphenylglycol (DHPG). These substances have been shown to be the major metabolites of norepinephrine in rat atria [16]. The homogenate was stored overnight at - 2 0 ° C then centrifuged at 5,000 rpm for 15 min. An aliquot of the supernatant (100 ul) was taken and spotted as a 3 cm line onto aluminium thin layer chromatography plates precoated with cellulose (Merck; layer thickness 0.1 mm). Spots were dried in cold air. The chromatograms were run for 4 hr in a solvent mixture n-butanol: 5 N acetic acid (100:35). The solvent system gave a good separation of all the components in question. The chromatograms were oven dried at 80°C for 15 min then sprayed with a mixture of 0.1 percent p-nitroaniline, 0.2 percent sodium nitrite, and 10 percent potassium carbonate in the ratio 1:1:2 in order to visualize the position of the standards. The TLC plates containing the homogenate and cold carriers were cut into segments containing the visualized spots and the region between the

NOREPINEPHRINE UPTAKE FOLLOWING STRESS

41 TABLE 1

THE ACCUMULATION OF RADIOACTIVITY IN ISOLATED SPONTANEOUSLY BEATING RAT ATRIA FOLLOWING INCUBATION WITH 3H_7.L.NOREPINEPHRINE

Uptake of Radioactivity (DPM/mg) mean _+ SE Incubation Time (min)

Control

2

t Test p

Stress

488 _+ 23 (10)

394 _+ 27

34 (12)

0.05

6.35

517 _+ 15 (5)

393 -+ 43 (5)